Team:Valencia UPV/Part Collection

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Assembly methods using type IIS restriction enzymes (based on the Golden Gate technology) have a great advantage over those using type II: the reaction is performed in a single step, without the need for gel band purification, therefore increasing the efficiency of the assembly and reducing the time consumed. In addition, the Golden Gate technology allows to assemble more than two pieces and the backbone in a single reaction. These characteristics allows the automation of the Golden Gate assembly method, what suits Printeria perfectly.

Despite these advantages of the Golden Gate technology, finding collections of parts in this standard optimized for the E. coli chassis and well characterized is very complicated. This is the reason why we decided to create our collection of basic parts, which is based on some of the most used parts in E. coli from the Registry of Standard Biological Parts.

To create it we used the GoldenBraid 3.0 standard, so they can be used both with the BioBrick and Golden Gate assembly methods . In addition, to give added value to this collection, we have made an exhaustive characterization of these parts.

Thus, we have included different types of basic parts so that, by combining them, we can create composite parts (transcriptional units) with different behaviors: constitutive or inducible, dependant of another genetic construction, single parts of genetic circuits such as oscillators, etc.

Basic Parts

All our basic parts are inserted in the plasmid BBa_P10500 and are compatible with BioBricks grammar, as they do not contain any illegal sites for RFC10. Moreover, they do not have sites for the type IIS restriction enzymes BsaI and BsmBI, used in the Golden Braid assembly. For information on how these parts have been designed, see our design page.


With respect to promoters, we include three constitutive promoters of different stregths, a strong promoter for T7 phage DNA polymerase, and a promoter for the sigma 32 subunit of E. coli DNA polymerase.

In addition, we have included three promoters regulated by a transcription factor: the inducible pBad, positively regulated by AraC in the presence of L-arabinose, and the inducible and repressible lux promoters, positively and negatively regulated by LuxR in the presence of Acyl-homoserine-lactone, respectively.

Table 1. Our promoter Collection.
Part Original BioBrick Description
BBa_K2656005 BBa_J23102 Constitutive promoter (relative strength 1)
BBa_K2656004 BBa_J23106 Constitutive promoter (relative strength 0.26)
BBa_K2656007 BBa_J23101 Constitutive promoter (relative strength 0.94)
BBa_K2656000 BBa_I719005 Strong promoter for T7 phage DNA polymerase
BBa_K2656001 BBa_K338001 Promoter for the sigma 32 subunit of E. coli DNA polymerase
BBa_K2656006 BBa_K2442101 pBad minimal promoter: positively regulated by AraC in the presence of L-arabinose
BBa_K2656002 BBa_R0061 Lux repressible promoter: negatively regulated by LuxR in the presence of Acyl-homoserine-lactone.
BBa_K2656003 BBa_R0062 Lux inducible promoter: positively regulated by LuxR in the presence of Acyl-homoserine-lactone.


Regarding the RBS, we have selected five from the Registry of Standard Biological Parts, with different strengths.

Table 2. Our RBS Collection.
Part Original BioBrick Description
BBa_K2656009 BBa_B0030 Strong RBS (relative strength 1)
BBa_K2656011 BBa_B0034 Medium RBS (relative strength 0.38)
BBa_K2656010 BBa_B0032 Weak RBS (relative strength 0.047)
BBa_K2656008 BBa_J61100 Very weak RBS (relative strength 0.042)
BBa_K2656012 BBa_J61101 Very weak RBS (relative strength 0.032)


As E. coli is our bacterial chassis, all these coding sequences have been codon optimized for this chassis organism.

We have chosen different coding sequences of pigmented proteins, both fluoroproteins (sfGFP, mRFP1, YFP and GFPmut3b) and chromoproteins (amilCP), as they are completely useful to design genetic constructions where there are necessary protein reporters of gene expression.

Moreover, another choice we have made is the addition of the LVA degradation tag to the mRFP1, YFP and GFPmut3b sequences, respectively. The LVA tag causes an increase in protease activity and, therefore, a faster degradation of the reporter protein in the cell. Thus, comparison between protein expression with and without the tag is a useful information to integrate in complex genetic oscillators design.

On the other hand, we have also selected the BSMT1 enzyme coding sequence, which will allow our bacteria to smell like mint in the presence of salicylic acid, to demonstrate odor gene expression.

Additionally, this collection also includes the transcriptional factors AraC and LuxR, that control the regulable promoters previously explained, as well as the CDS of LuxI, that acts producing the Acyl-Homoserine-Lactone to which LuxR responds. For this last CDS, we had to codon optimize its sequence for E. coli.

Finally, we have picked a lysis gene from the enterobacteria phage phiX174, which allows us to create genetic circuits which functionality implies the death of part of the population.

Thus, all these coding sequences are a toolkit of useful basic pieces to supply the user with the necessary pieces to construct the basis of common bacterial genetic circuits.

Table 3. Our CDS Collection.
Part Original BioBrick Description
BBa_K2656013 BBa_I746916 Superfolder Green fluorescent Protein (sfGFP) coding sequence
BBa_K2656014 BBa_E1010 monomeric Red fluorescent protein (mRFP1) coding sequence
BBa_K2656021 BBa_K592101 Yellow fluorescent protein (YFP) coding sequence
BBa_K2656022 BBa_E0040 Green fluorescent protein (GFPmut3b) coding sequence
BBa_K2656018 BBa_K592009 amilCP (Blue chromoprotein) coding sequence
BBa_K2656024 BBa_K1399001 mRFP1 with the LVA tag coding sequence
BBa_K2656020 None YFP with the LVA tag coding sequence
BBa_K2656023 BBa_K1399004 GFPmut3b with the LVA tag coding sequence
BBa_K2656025 BBa_J45004 BSMT1 coding sequence
BBa_K2656017 BBa_K2442103 AraC transcription factor coding sequence
BBa_K2656016 BBa_C0062 LuxR transcription factor coding sequence
BBa_K2656019 BBa_C0161 LuxI (Acyl-Homoserine-Lactone synthase) codon optimized coding sequence
BBa_K2656015 None Lysis gene from the enterobacteria phage phiX174

Transcriptional terminators

Lastly, as far as terminators are concerned, we have only chosen one, as we do not think that introducing several would have an influence on the functioning of Printeria. The terminator chosen is B0015 because it is the most used in E. Coli.

Table 4. Our Transcriptional Terminator.
Part Original BioBrick Description
BBa_K2656026 BBa_B0015 Double transcriptional terminator

Composite Parts

In order to make the necessary measurements to characterize the basic parts and demonstrate that they are functional, we have built some composite parts by combining a promoter, an RBS, a CDS and the terminator in a GoldenBraid Alpha 1 vector with the Golden Gate assembly protocol .


With Printeria, one of our main goals is to promote the Microbial Art among society. Thus, we decided to provide the bioartists with a complete ART DNA toolkit for its use with Printeria. As they use living cells as primary material for their artworks, we wanted to produce an extense palette: Printone.

Printone is composed of a colection of transcriptional units assembled with each of our fluorescent and coloured basic parts. By changing the promoters and RBS strengths, a wide range of different tonalities were obtained. Moreover, the LVA tag allowed us to reduce the brightness and so include degraded tones.

Image 1. Our Printeria colors: Some of the Printone transcriptional units, with variable expression levels of mRFP, YFP and amilCP proteins
Table 5. Our BioArt DNA toolkit palette: Printone.
Transcriptional unit Intensity CDS
BBa_K2656105 Medium: Low promoter, strong RBS GFPmut3b
BBa_K2656106 Medium: low promoter, strong RBS GFPmut3b
BBa_K2656107 Strong: Strong promoter, strong RBS GFPmut3b
BBa_K2656117 Low: Low promoter, medium RBS GFPmut3b
BBa_K2656108 Degradation tag: less colour intensity GFPmut3b+LVA tag
BBa_K2656101 Medium: Low promoter, strong RBS sfGFP
BBa_K2656100 Low: weak promoter, very weak RBS sfGFP
BBa_K2656102 Low: weak promoter, weak RBS sfGFP
BBa_K2656103 Medium: weak promoter, medium RBS sfGFP
BBa_K2656104 Very low: weak promoter, very weak RBS sfGFP
BBa_K2656109 Medium: Low promoter, strong RBS mRFP1
BBa_K2656118 Low: Low promoter, weak RBS mRFP1
BBa_K2656119 Medium: Low promoter, medium RBS mRFP1
BBa_K2656110 Degradation tag: less colour intensity mRFP1+LVA tag
BBa_K2656113 Medium: Low promoter, strong RBS amilCP
BBa_K2656120 Medium: Low promoter, medium RBS amilCP
BBa_K2656112 Medium: Low promoter, strong RBS YFP
BBa_K2656111 Degradation tag: less colour intensity YFP+LVA tag

Transcriptional units: complete table

This DNA Part Collection also includes non- constitutive transcriptional units and TU assemblies related with genetic circuits regulation, such as the HSP-GFPmut3b transcriptional unit, pLuxR-GFPmut3b transcriptional unit, LuxI constitutive transcriptional unit or AraC constitutive transcriptional unit.
Table 6. Our composite part complete collection
Composite Part PromoterRBSCDSTerminatorDescription
BBa_K2656100BBa_K2656004BBa_K2656008BBa_K2656013BBa_K2656026sfGFP transcriptional unit 1
BBa_K2656101BBa_K2656004BBa_K2656009BBa_K2656013BBa_K2656026sfGFP transcriptional unit 2
BBa_K2656102BBa_K2656004BBa_K2656010BBa_K2656013BBa_K2656026sfGFP transcriptional unit 3
BBa_K2656103BBa_K2656004BBa_K2656011BBa_K2656013BBa_K2656026sfGFP transcriptional unit 4
BBa_K2656104BBa_K2656004BBa_K2656012BBa_K2656013BBa_K2656026sfGFP transcriptional unit 5
BBa_K2656105BBa_K2656004BBa_K2656009BBa_K2656022BBa_K2656026GFP transcriptional unit 1
BBa_K2656106BBa_K2656005BBa_K2656009BBa_K2656022BBa_K2656026GFP transcriptional unit 2
BBa_K2656107BBa_K2656007BBa_K2656009BBa_K2656022BBa_K2656026GFP transcriptional unit 3
BBa_K2656108BBa_K2656004BBa_K2656009Bba_K2656023BBa_K2656026GFP_LVA transcriptional unit
BBa_K2656109BBa_K2656004BBa_K2656009Bba_K2656014BBa_K2656026mRFP1 transcriptional unit 1
BBa_K2656110BBa_K2656004BBa_K2656009Bba_K2656024BBa_K2656026mRFP1_LVA transcriptional unit 1
BBa_K2656111BBa_K2656004BBa_K2656009BBa_K2656020BBa_K2656026YFP_LVA transcriptional unit 1
BBa_K2656112BBa_K2656004BBa_K2656009BBa_K2656021BBa_K2656026YFP transcriptional unit 1
BBa_K2656113BBa_K2656004BBa_K2656009BBa_K2656018BBa_K2656026amilCP transcriptional unit 1
BBa_K2656114BBa_K2656004BBa_K2656009BBa_K2656016BBa_K2656026Constitutive LuxR transcriptional unit
BBa_K2656115BBa_K2656001BBa_K2656009BBa_K2656022BBa_K2656026HSP-GFPmut3b transcriptional unit
BBa_K2656116BBa_K2656002BBa_K2656009BBa_K2656022BBa_K2656026pLuxR-GFPmut3b transcriptional unit
BBa_K2656117BBa_K2656004BBa_K2656011BBa_K2656022BBa_K2656026GFPmut3b transcriptional unit 4
BBa_K2656118BBa_K2656004BBa_K2656010BBa_K2656014BBa_K2656026mRFP transcriptional unit 2
BBa_K2656119BBa_K2656004BBa_K2656011BBa_K2656014BBa_K2656026mRFP transcriptional unit 3
BBa_K2656120BBa_K2656004BBa_K2656011BBa_K2656018BBa_K2656026amilCP transcriptional unit 2
BBa_K2656121BBa_K2656004BBa_K2656011BBa_K2656017BBa_K2656026AraC constitutive transcriptional unit
BBa_K2656122BBa_K2656003BBa_K2656011BBa_K2656022BBa_K2656026pLux-GFPmut3b transcriptional unit
BBa_K2656123BBa_K2656000BBa_K2656010BBa_K2656022BBa_K2656026pT7-GFPmut3b transcriptional unit
BBa_K2656124BBa_K2656000BBa_K2656011BBa_K2656022BBa_K2656026pT7-GFPmut3b transcriptional unit 2
BBa_K2656125BBa_K2656004BBa_K2656009BBa_K2656019BBa_K2656026LuxI constitutive transcriptional unit 1
BBa_K2656126BBa_K2656004BBa_K2656009BBa_K2656025BBa_K2656026BMSTI transcriptional unit


Our transcriptional units were built using a GoldenBraid 3.0 alpha 1 vector which is not compatible with the BioBrick grammar. Therefore, we have created the BBa_K2656200 plasmid, adapted from the standard pSB1C3 backbone, to convert our GoldenBraid transcriptional units to BioBricks.

BBa_K2656200 is a modified version of BBa_P10500, so it includes BsmBI restriction sites flanking the selection marker. When digesting this plasmid with BsmBI, the result is complementary sticky ends to those formed in a GoldenBraid alpha 1 plasmid when digesting with the same enzyme. By this way, the BioBrick compatibility for each composite part is easily achieved using this plasmid as the destination vector in a Golden Gate BsmBI one-pot reaction.

Screening marker

Using the BBa_P1050 plasmid, selection of positive transformants relies on the white-blue screening method, which is based on the α-complementation phenomena. To do so, the host strain must carry the ω-peptide, while the vector contains the α-peptide. If both subunits are expressed (when there is not insertion in the vector), functional beta-galactosidase is reconstituted. Thus, blue-white screening is carry out by the addition of X-gal (D-lactose analog) and IPTG into the agar plates. In this way, if there is no insertion, colonies will be blue as a consequence of the X-gal hidrolysis. Although the principle is simple, this method requires the use of lacZ mutant strains and it is always necessary to add both reagents into the medium.

In order to avoid these limitations, we have decided to rely on a different visual method. Thus, we have inserted an mRFP1 transcriptional unit (BBa_K2656201) to act as the screening gene marker. By this way, cloning is achieved in a more reliable, efficient, low-cost way, so avoiding the necessity of adding chemical supplements and the possible non-function of these substances once on the solid medium. With this method, non-positive transformants will always express an intense red fluorescent protein, while non-recombinant transformants will not be able to express this protein.

Table 7. Our designed plasmids
Part Description
BBa_K2656200 BioBricks compatible plasmid